Fractionating/collecting device of liquid chromatograph

ABSTRACT

In a preferred embodiment, a sample container storage part for storing a number of sample containers S, a nozzle for dropping a sample component separated and supplied by an LC and an additive liquid such as digestive fluid supplied from another liquid supplying part to the sample container S, a carrying mechanism for carrying and positioning the sample container at an arbitrary position under the nozzle, and a second nozzle, serving as a suction/injection mechanism, for sucking in the fractionated/collected sample component and injecting the sample component to another LC. The carrying mechanism provides a rotation mechanism. The carrying mechanism rotates over 180 degrees and carries the sample container S completed with fractionating/collecting to the position of the second nozzle, and the sample is sucked in by the second nozzle and injected to the LC of next stage.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to fractionating/collecting devices forfractionating and collecting a separated sample component in a liquidchromatograph, hereinafter also referred to as LC, performing separationanalysis of a solution sample.

2. Description of the Related Art

An effluent from a liquid chromatograph is fractionated and collected,and further analyzed in the liquid chromatograph or other analyzingdevices such as a mass spectrograph (MS). There afractionating/collecting device for collecting onto a sample containersuch as a microplate is used.

One example of the mass spectrograph for analyzing the effluent from theliquid chromatograph is a Matrix Assisted LaserDesorption/Ionization-Time of Flight Mass Spectrometry (MALDI-TOFMS). Inthe MALDI-TOFMS, when fractionating and collecting sample componentsseparated in the liquid chromatograph on the MALDI-TOFMS analyzing plateusing the fractionating/collecting device, the sample components arecollected at a number of sites on a single plate. After the collectingprocess is completed, the plate is manually moved to the MALDI-TOFMSdevice for analysis.

As only one or two collecting plates can be used in the conventionalfractionating/collecting devices, not many analyzing samples can beprepared at night without any workers present, for example, for suchMALDI-TOFMS analysis.

Other analyzing methods for analyzing the effluent from the liquidchromatograph includes an LC for performing separation analysis throughmultiple stages, which include fractionating and collecting the samplecomponent separated in the LC of the first stage with thetractionating/collecting device, and analyzing the collected samplecomponent in the LC of the second stage after processing. In theanalysis of protein, for example, in order to further analyze theprotein separated in the LC, a digestive enzyme is manually added to thedivided and collected sample component of the protein for decomposing topeptide, the peptide being further separated in another LC for analyzingin the MS, and analysis is carried out on the obtained MS data.

In the two-step separation task in the LC, manual tasks such as addingthe digestive enzyme take a long time. Therefore, a liquid handlercapable of fractionating/collecting and dispensing is used forautomatically carrying out the task. Normally in the liquid handler, itsprobe moves to a fractionating/collecting position of the LC in thefirst stage to suck in the collected component, and then moves to aninjection port in the LC of the second stage to inject the suckedcomponent Thus, the space in the probe is large, which isdisadvantageous especially when a micro LC, for example, is used on thefractionating/collecting side to handle very small amounts of sample.

SUMMARY OF THE INVENTION

The first object of the present invention is to propose afractionating/collecting device capable of collecting samples on anumber of sample containers.

The second object of the present invention is to further propose afractionating/collecting device capable of shortening time and savinglabor in handling the fractionated components between the first andsecond stages in a LC and which is capable of analyzing a small amountof fractionated component.

The fractionating/collecting device according to the present inventionfor achieving the first object comprises a fractionating/collectingmechanism for dropping and collecting an effluent from a liquidchromatograph to a sample container from a nozzle, a storage part forstoring a number of sample containers, and a carrying mechanismcomprising a vertical moving mechanism and a horizontal moving mechanismfor both carrying and positioning the sample container to an arbitraryposition under the nozzle.

In one aspect of the fractionating/collecting device of the presentinvention, multiple samples to be analyzed can be prepared since samplecollecting can be carried out on a number of sample containers and thesample containers can be stored by providing the storage part forstoring a number of sample containers and by configuring the carryingmechanism so as to rotate within a range of the device and to movefreely in the vertical and horizontal directions. Furthermore, thecarrying mechanism is also used to position the sample container duringcollecting, and thus the device can be inexpensively realized.

Continuous analysis can also be carried out by combining theconventional fractionating/collecting device and a multi-purpose robotarm. However, in comparison with a device of the present invention, suchrobot arm does not only require to be a two-system robot arm whichincludes a carrying mechanism for carrying the plate and a collectionaiding mechanism for positioning the plate so that a sample liquid isdropped onto an appropriate position on the plate under the nozzledropping the effluent from the LC, but also requires a large area toinstall the robot arm.

If the nozzle is configured so that the effluent from the liquidchromatograph and a liquid from another liquid supplying part aresimultaneously dropped a matrix liquid, for example, and the effluentfrom the liquid chromatograph may be simultaneously dropped to form thedevice for preparing a sample for MALDI-TOFMS exclusively.

If the nozzle is configured so that either the effluent from the liquidchromatograph or a liquid from another liquid supplying part isselectively dropped only the effluent from the liquid chromatograph, forexample, may be dropped, and a desired liquid such as matrix liquid ordigestive fluid may be subsequently selectively dropped to form amultipurpose sample preparing device.

If the carrying mechanism is configured to be able to carry the samplecontainer, in which sample collecting is completed, to a predeterminedposition out of the fractionating/collecting device, the samplecontainer can not only be stored within the storage part but can also beautomatically loaded to an analyzing device.

If an outlet for discharging gas in the direction of the tip portion ofthe nozzle is provided at the periphery of the tip portion of thenozzle, it can prevent liquid from remaining at the tip portion of thenozzle.

When a sample fraction dropped onto the plate is dried and crystallized,crystallization will not be even if the moisture content within thecollecting device under analyzing process changes. A port for exhaustinggas provided in the vicinity of the nozzle can stabilize drying of thefraction.

A fractionating/collecting device of the present invention for achievingthe second object comprises a fractionating/collecting mechanism fordropping an effluent from liquid chromatograph to a sample containerwith a nozzle, a carrying mechanism comprising a vertical movingmechanism and a horizontal moving mechanism for both carrying andpositioning the sample container to an arbitrary position under thenozzle, a sample container storage part provided within a movable rangeof the sample container by the carrying mechanism for storing a numberof sample containers, and a suction/injection mechanism provided withina movable range of the sample container by the carrying mechanism forsucking in a sample in the sample container and injecting the suckedsample to another LC.

In another aspect of the present invention, as thefractionating/collecting device comprises a carrying mechanism forcarrying and positioning the sample container, and the mechanism forcarrying out suction and injection of samples for LC in the next stagewithin the movable range of the sample container, the LC provided withsuch fractionating/collecting device can carry out multi-stagechromatograph separation without any manual aid inexpensively andautomatically. Furthermore, automatization increases the rate ofoperation.

As the movable range of the suction/injection mechanism can berestricted by moving the sample container, dead space in the nozzlemechanism of the suction/injection mechanism can be decreased and verysmall amounts of fraction component can be handled.

The fractionating/collecting device of an open space type (non-closedconfiguration) has a risk of allowing a condensed water into the samplecontainer when, for example, the sample is being cooled down and thus itis not an ideal condition. If the fractionating/collecting mechanism,the carrying mechanism, the sample container storage part, and thesuction/injection mechanism are stored within a sealed space providedwith a temperature control mechanism, it is advantageous for preventingcondensation when the sample is being cooled down.

If the nozzle for dropping the effluent from the LC is connected to aliquid supplying part for dropping another liquid, for example, anadditive liquid such as digestive fluid simultaneously with the drop ofthe effluent, a device used exclusively for sample preparation tore-separate the fractionated/collected sample can be obtained.

If the nozzle for dropping the effluent from the LC is connected to aliquid supplying part for arbitrary selecting and dropping anotherliquid at a different timing with the drop of the effluent, amultipurpose sample preparation device for adding an arbitrary additiveliquid to the fractionated/collected sample can be obtained.

Other aspects and advantages of the invention will become apparent fromthe following description, taken in conjunction with the accompanyingdrawings, illustrating by way of example the principles of theinvention.

BRIEF DESCRIPTION OF THE DRAWINGS

The invention, together with aims and advantages thereof, may best beunderstood by reference to the following description of the presentlypreferred embodiments together with the accompanying drawings in which:

FIG. 1 is a perspective view showing a configuration of one embodimentof the present invention;

FIG. 2 is a cross sectional view showing a nozzle and mechanisms at theperiphery of the nozzle of the embodiment in FIG. 1; and

FIG. 3 is a schematic diagram showing a configuration according toanother embodiment of the present invention.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

FIG. 1 is a schematic diagram showing a preferred embodiment of thepresent invention.

In FIG. 1, a fractionating/collecting device includes a carryingmechanism 3 arranged next to a mass spectrograph 12 for carrying andpositioning a sample container, and a storage part 6 arranged next tothe carrying mechanism 3 for storing a number of sample containers. Thefractionating/collecting device further includes a nozzle 22 arranged ata position not in the way of the carrying mechanism 3 for discharging amatrix liquid and an effluent from a liquid chromatograph, a port 20arranged in the vicinity of the nozzle 22 for supplying dry air, and atube 18 arranged at a periphery of a tip portion of the nozzle 22 forexhausting air in the direction of the tip portion. The tube 18 isconnected to a syringe (not shown).

The storage part 6 has shelves for storing a number of samplecontainers, and is able to store sample containers in which the samplecollecting is incomplete and the sample containers in which the samplecollecting is complete. The sample containers are, for example, for aMALDI-TOFMS sample plates.

The carrying mechanism 3 includes a holder 4 for holding the samplecontainer, an X-R stage 2 which is provided with a driving mechanism formoving the holder 4 within a plane (X direction), and rotatable (Rdirection) and movable in the vertical direction (Z direction), amechanism (not shown) for supporting the X-R stage 2, a rod screw 8 formoving the X-R stage 2 in the vertical direction, a Y stage 10, movablein the Y direction whithin a plane, for supporting the rod screw 8 and aguide 16 for moving the Y stage 10 in the Y direction.

A supporter 30 of the X-R stage 2 has a screw that fits with the rodscrew 8. As the rod screw 8 rotates, the X-R stage 2 moves in thevertical direction.

A bearing 32 is provided to the supporter 30, allowing the X-R stage 2to be maintained in a predetermined direction independently from therotation of the rod screw 8 and to rotate within the plane.

The Y stage 10 is provided with a mechanism for moving in the Ydirection along the guide 16, and a mechanism for rotating the rod screw8.

The sample container held to be carried by the carrying mechanism 3 canbe moved to and positioned at an arbitrary position within the movablerange by the moving of the holder 4 in the X direction, the verticalmoving as well as the rotation of the X-R stage 2, and the moving of theY stage 10 in the Y direction.

FIG. 2 shows the nozzle 22 for discharging the liquid and mechanisms inthe periphery thereof of the above embodiment.

A tube 26 for introducing the effluent from the liquid chromatograph anda tube 24 for introducing the matrix liquid are connected to the nozzle22. The tube 18 for introducing air is arranged at the periphery of thenozzle 22. Since the nozzle 22 discharges very small amounts of liquid,the liquid sometimes remains at the tip portion of the nozzle 22 due tothe surface tension, and can not be satisfactorily dropped onto thesample container. The air exhausted from the periphery of the tipportion of the nozzle 22 then stimulates the liquid to drop.

The port 20 arranged in the vicinity of the nozzle 22 for exhausting dryair blows the dry air to the sample liquid dropped onto the samplecontainer and dries the sample liquid.

The operation of the present embodiment will now be described.

First, the Y stage 10 of the carrying mechanism 3 is moved to apredetermined position of the storage part 6, the rod screw 8 is rotatedto move the X-R stage 2 in the Z direction for adjustment of height, theholder 4 is moved in the X direction from the X-R stage 2 to take outthe predetermined sample container.

After the sample container is taken out, the carrying mechanism 3 movesthe sample container to under the nozzle 22.

Under the nozzle 22, in order to collect the effluent from the liquidchromatograph and the matrix liquid dropped from the nozzle 22 at apredetermined position on the sample container, the carrying mechanism 3is fine-adjusted in the X and Y directions to be positioned at theliquid drop. After the positioning is finished, the liquid is dropped.The air exhausted from the tube 18 from the air discharging syringestimulates the liquid dropping.

When the liquid has been dropped, the dry air is blown from the port 20,and the dropped sample liquid is dried and crystallized.

The positioning, dropping and the drying processes are repeatedlycarried out until the fractionating/collecting of the relevant samplecontainer is complete.

After fractionating/collecting is completed, the carrying mechanism 3carries the sample container to the storage part 6 again to store thesample container in the storage part 6. Then the carrying mechanism 3takes out a new sample container, carries the sample container to underthe nozzle 22, and repeats the fractionating/collecting process.

By repeating such processes, a number of sample containers in whichfractionating/collecting is complete are stored in the storage part 6.

When analyzing by mass spectrometry, the holder 4 takes out the samplecontainer in which fractionating/collecting is complete from the storagepart 6, and the X-R stage 2 rotates over 180 degrees to move the samplecontainer to a loading part 14 of the mass spectrograph 12 for loading.

The sample container in which the sample fractionating/collecting iscomplete may also be directly loaded from under the nozzle 22 to themass spectrograph 12.

FIG. 3 is a schematic diagram showing a configuration of anotherembodiment.

The fractionating/collecting device of this embodiment is provided witha sample container storage part 6 capable of storing a number of samplecontainers S such as a microplate, a nozzle 40 for dropping a samplecomponent separated and supplied by the LC and an additive liquid suchas digestive fluid supplied from other liquid supplying part to thesample container S, a carrying mechanism 3 for carrying and positioningthe sample container S at an arbitrary position under the nozzle 40, anda nozzle 60, which serves as a suction/injection mechanism, for suckingin the fractionated/collected sample component and injecting the samplecomponent to another LC in a housing F shown with a dashed line. Thehousing F has a temperature control function to maintain its internalspace at a predetermined temperature, and has a sealed configuration inwhich the internal space is externally blocked.

The sample container storage part 6 has shelves for storing a number ofsample containers, and both the sample container in which the samplecollecting is incomplete and the sample container in which the samplecollecting is complete can be stored.

The carrying mechanism 3 is the same as that shown in FIG. 1 and thusthe description thereof shall be omitted.

The nozzle 40 is arranged in the vicinity of the carrying mechanism 3 soas not to be in the way of the sample container storage part 6. A tubefor sending the effluent from the LC, and a tube for sending an additiveliquid 52 such as digestive enzyme or a reactive liquid are connected tothe nozzle 40.

In the LC, an eluate 50 is supplied by a pump 48 and sends a samplewhich is injected from an injector 46, to a column 44. The sampleseparated in the column 44 is detected in a detector 42 and is droppedfrom the nozzle 40 and fractionated/collected at the sample container S.

Furthermore, the additive liquid 52 is supplied by a pump 49, and isdropped from the nozzle 40 to the sample container S. The additiveliquid 52 can be dropped onto the sample container S simultaneously withthe effluent from the LC by having the pump 49 continuously supplied theliquid, or can be dropped after the effluent from the LC is dropped byhaving the pump 49 supplied the liquid over a predetermined time,.

The nozzle 60 is arranged within a movable range of the sample containerS at a position different from the nozzle 40. In this embodiment, thenozzle 60 is arranged on the side opposite the nozzle 40 with respect tothe carrying mechanism 3. The nozzle 60 is movably supported so as to beable to suck in the sample in the sample container S and inject thesucked sample into an injection port 54 placed within the housing F.

The injection port 54 is connected to an injector 46 a of the LC of thenext stage, and injects the sample to the LC of the next stage by way ofthe injector 46 a. In the LC of the next stage, the eluate 52 a issupplied by a pump 48 a, and sends the sample, which is injected fromthe injector 46 a, to a column 44 a. The sample component separated inthe column 44 a is supplied to the MS, which is acting as a detector, tobe detected.

The operation of the present embodiment shall now be described.

First, the carrying mechanism 3 moves to the position of the samplecontainer storage part 6, and the holder 4 moves in the X direction totake out the sample container S. The sample container S taken out iscarried to under the nozzle 40, and the effluent from the LC and theadditive liquid. 52 such as digestive enzyme are dropped thereon fromthe nozzle 40. The collecting task is carried out by positioning thesample container in the X and Y directions by means of the carryingmechanism 3 so that the effluent 50 and the additive liquid 52 aredropped to an appropriate location on the sample container S.

The sample container S, in which the sample collecting is complete, isstored in the sample container storage part 6, a new sample container Sis taken out from the sample container storage part 6, and thecollecting of the new sample is repeated. In the sample containerstorage part 6, the collected samples are maintained at a constanttemperature.

The sample container S completed with sample collecting and stored inthe sample container storage part 6 is taken out as needed from thesample container storage part 6 by means of the carrying mechanism 3.Then the X-R stage 2 is rotated over 180 degrees with the rod screw 8 asthe center and is further adjusted in height to position the samplecontainer S under the nozzle 60. The nozzle 60 sucks in the sample inthe sample container S, moves to the injection port 54 placed nearby andinjects the sucked sample to the injection port 54.

The sample injected to the injection port 54 is sent to the column 44 awith the eluate 52 a supplied by the pump 48 a from the injector 46 a,and is further separated and sent to the MS for detection.

1. A fractioning/collecting device comprising: a fractioning/collectingmechanism for dropping and collecting an effluent from liquidchromatograph onto a sample container with a nozzle; a carryingmechanism providing a vertical moving mechanism and a horizontal movingmechanism for carrying and position the sample container at an arbitraryposition under the nozzle; a sample container storage part arrangedwithin a movable range of the sample container by the carrying mechanismfor storing a number of sample containers; and a suction/injectionmechanism arranged within a movable range of the sample container by thecarrying mechanism for sucking in a sample in the sample container andinjecting the sucked sample into another liquid chromatograph.
 2. Thefractioning/collecting device according to claim 1, wherein thefractioning/collecting mechanism, the carrying mechanism, the samplecontainer storage part and the suction/injection mechanism are arrangedin a sealed space provided with a temperature control mechanism.
 3. Thefractioning/collecting device according to claim 1, wherein the nozzleis connected to a liquid supplying part for dropping a different liquidsimultaneously with the dropping of the effluent from the liquidchromatograph.
 4. The fractioning/collecting device according to claim1, wherein the nozzle is connected to a liquid supplying part fordropping a different liquid arbitrarily selected at a different timingwith the effluent from the liquid chromatography.